Dr. Stanley and his colleagues have discovered a new disorder of hypoglycemia in infants who have an unusual combination of congenital hyperinsulinism together with a persistent hyperammonemia. Their preliminary results demonstrate that the site of defect in this hyperinsulinism/hyperamonemia (HI/HA) syndrome is the mitochondrial enzyme, glutamate dehydrogenase (GDH). In this novel inborn error of intermediary metabolism, affected patients have mutations of GDH that are dominantly expressed and result in a gain, rather than a loss, of enzyme function. The mutations in some, (HI/HA) patients are located in a GTP inhibitory allosteric domain of GDH. Their discovery of these GDH mutations in (HI/HA) patients indicates that GDH plays a central role in the regulation of beta-cell insulin secretion and hepatic ammonia detoxification which has not previously been recognized. The goals of this grant are to understand the mechanisms by which the HI/HA mutations of GDH affect the control of enzyme function and subsequently lead to dysregulation of amino acid and glucose metabolism. The hypothesis is that, because of impaired allosteric control, GDH mutations cause excessive oxidation of glutamate which in turn, in appropriately promotes insulin secretion by pancreatic beta-cells while simultaneously depleting the high concentrations of glutamate in liver that are necessary to activate ureagenesis. Aim 1 is to determine the location of GDH mutations that can cause the HI/HA syndrome and to correlate these genotypes with differences in clinical phenotypes and patterns of abnormality in GDH enzymatic activity from cultured patient lymphoblasts. Aim 2 is to characterize the effect of different GDH mutations on the catalytic activity and allosteric responsiveness of purified mutant enzymes expressed in E. coli. Aim 3 is to demonstrate the effects of GDH mutations on the control of glutamate oxidation and glutamate concentrations in intact cells with the use of cultured lymphoblasts from HI/HA patients. Aim 4 is to define the mechanism by which GDH mutations of HI/HA patients lead to abnormal regulation of insulin secretion in beta-cells and ammonia detoxification in liver using transgenic mice expressing mutant enzyme specifically in islets or liver.